Regulation of acinar cell function

腺泡细胞通过调控胞吐作用产生、储存和分泌酶，该过程涉及对刺激的空间和时间上的钙离子释放。控制钙离子浓度([Ca~(2+)])对基因转录和蛋白质加工也是至关重要的。不适当的[Ca~(2+)]会改变基因的表达，影响蛋白质的加工，促进细胞凋亡。最近，我们发现了一种新的分泌途径Ca~(2+)-ATPase-2(SPCA2)亚型，称为SPCA2C，它与较长的SPCA2蛋白的羧基末端一致，但缺乏ATPase结构域。SPCA2C过表达增加内质网和胞浆[Ca~(2+)]，并影响G蛋白偶联受体(GPCR)信号和存储操作的Ca~(2+)内流(SOCE)。SOCE在内质网低[Ca~(2+)]时被激活，激活STIM和ORAI通道，将细胞外的Ca~(2+)输送到内质网。本研究下一个周期的短期目标是：(1)确定SPCA2C影响腺泡和非腺泡细胞内钙稳态的机制，以及(2)确定与SPCA2C相互作用的蛋白质以调节其功能。管理假设：SPCA2C是腺泡细胞功能所必需的钙稳态的重要调节因子。AIMS：(1)确定受SPCA2C影响的钙信号通路。标记的SPCA2C(SPCA2CFLAG)将在表达YFP标记的Orai1(HEK293-Orai1YFP)的HEK293或HEK293细胞中表达。将研究GPCR信令、SOCE和存储独立的钙离子进入(SICE)。FURA2成像、共聚焦显微镜和共聚焦显微镜将评估细胞内游离钙浓度([Ca~(2+)]i)和蛋白质定位。SOCE和GPCR信号通路的药物抑制剂将决定SPCA2C在这些通路中的作用。(2)确定SPCA2C在腺泡细胞生物学中的作用。我们已经开发了腺病毒载体，用于在腺泡细胞培养或体内通过导管内注射表达表位标记的SPCA2C。我们将评估SPCA2C过表达后的腺泡细胞形态、[Ca+]i、基因表达和胞吐能力，以确定其在腺泡细胞中对这些功能的重要性。(3)确定SPCA2C的蛋白质相互作用因子。我们将使用BioID进行无偏筛选，然后进行质谱分析，以确定SPCA2C的相互作用蛋白。我们将使用co-IP和Lumiere分析来验证HEK293-Orai1YFP细胞和腺泡细胞中SPCA2C的相互作用伙伴。识别的蛋白质的特征将如AIMS 1和2中所述，这些实验将定义SPCA2C相互作用的蛋白质，其也可能介导[Ca~(2+)]。表征SPCA2C将解决我们的长期目标：(A)描述不同的Ca~(2+)调节信号通路被协调以维持腺泡功能和响应环境提示的机制，以及(B)定义这些通路的变化如何影响关键的细胞活动，包括转录、蛋白质折叠和胞吐作用。

Acinar cells produce, store and secrete enzymes through a process known as regulated exocytosis, which involves spatial and temporal Ca2+ release in response to stimulation. Control of Ca2+ concentrations ([Ca2+]) is also essential for gene transcription and protein processing. Improper [Ca2+] alters gene expression, affects protein processing and promotes apoptosis. Recently, we identified a novel isoform of Secretory Pathway Ca2+ATPase 2 (SPCA2) termed SPCA2C, which aligns with the carboxy terminus of the longer SPCA2 protein, but lacks the ATPase domain. Over-expression of SPCA2C increases ER and cytosolic [Ca2+] and affects G protein coupled receptor (GPCR) signalling and Store-Operated Ca2+ Entry (SOCE). SOCE is activated in response to low [Ca2+] in the ER, which activates STIM and ORAI channels to transport Ca2+ into the ER from outside the cell. The short term goals for the next cycle of this research program are to (1) determine mechanisms by which SPCA2C affects Ca2+ homeostasis in acinar and non-acinar cells, and (2) identify proteins that interact with SPCA2C to regulate its function.Governing hypothesis: SPCA2C is an important regulator of Ca2+ homeostasis essential for acinar cell function.AIMS:(1) Identify Ca2+-signaling pathways affected by SPCA2C. Tagged SPCA2C (SPCA2CFLAG) will be expressed in HEK293 or HEK293 cells expressing YFP-tagged Orai1 (HEK293-Orai1YFP). GPCR signalling, SOCE and Store Independent Ca2+ Entry (SICE) will be examined. Fura2 imaging, co-IF, and confocal microscopy will assess cytosolic free Ca2+ concentration ([Ca2+]i) and protein localization. Pharmacological inhibitors of SOCE and GPCR signaling will determine SPCA2C's role in these pathways.(2) Identify the role of SPCA2C on acinar cell biology. We have developed adenoviral vectors for expression of epitope-tagged SPCA2C in acinar cells cultures or in vivo through intraductal injection. We will assess acinar cell morphology, [Ca2+]i, gene expression and exocytosis ability following SPCA2C over-expression to determine its importance to these functions in acinar cells.(3) Define the protein interactors for SPCA2C. We will perform an unbiased screen using BioID followed by mass spectrometry to identify interacting proteins for SPCA2C. We will use co-IP and Lumiere assays to validate interacting partners for SPCA2C in HEK293-Orai1YFP cells and acinar cells. Identified proteins will be characterized as described in Aims 1 and 2, These experiments will define SPCA2C interacting proteins which may also mediate [Ca2+]i.Characterizing SPCA2C will address our Long Term Goals to (a) delineate mechanisms by which diverse Ca2+-regulating signaling pathways are coordinated to maintain acinar function and respond to environmental cues, and (b) define how changes to these pathways affect critical cell activities including transcription, protein folding and exocytosis.